DNA POLYMERASE

DNA POLYMERASE
DNA polymerases are a group of enzymes required for DNA synthesis.
These enzymes make new copies of DNA from existing templates and
also function by repairing the synthesized DNA to prevent mutations.
DNA polymerase catalyzes the formation of the phosphodiester bond
which makes up the backbone of DNA molecules.
Arthur Kornberg in 1956. purified and characterized DNA polymerase
from E.coli for the first time.
It is a single-chain polypeptide now known as DNA polymerase-I. 
now found five DNA polymerases in E. coli.
DNA POLYMERASE
DNA polymerases are not used for initiating the synthesis of
new strands, but in the extension of already existing DNA or
RNA strands which are paired with a template strand.
Some types of DNA polymerase have the ability to proofread
and remove unmatched bases of nucleotides and correct them.
They also correct post-replication mismatches by monitoring
and repairing the errors, by distinguishing mismatches of the
new strand from the template strand sequences.
DNA POLYMERASE STRUCTURE
1. The DNA polymerases generally have a conserved structure, and therefore,
defining its vital role in the cell function which can not be replaced.
2. DNA polymerases are made up of subdomains resembling an open right hand
as palm, fingers, and thumb.
3. The palm contains the catalytic essential amino acids in its active sites.
4. The fingers play a major role in nucleotide recognition and binding.
5. The thumb is for binding of the DNA substrate
6. There is a domain found between the finger and the thumb known as a
pocket, which is made up of two regions i.e the insertion site and the post-
insertion site.
7. The incoming nucleotides bind to the insertion site while the new base pair
bind in the post-insertion site.
Structure of Family A
 Family A polymerase additionally has a 5′ to 3′ exonuclease which is used to remove the RNA primers
from the Okazaki fragments.
 Some Family A groups also have a 3′ to 5′ exonuclease activity which functions in proofread the DNA.
Structure of Family B
 They also possess the basic subdomains with extremely active 3′ to 5; exonuclease to correct the errors
of DNA replication.
Structure of Family X
 These family groups have the thumb, palm, and finger subdomains which are structurally part of the N-
terminal or on the 31-kDA polymerase fragment.
 The N-terminal is connected to an 8kDa amino-terminal domain which contains a 5′ deoxyribose
phosphate lyase, which is essential in base excision repair.
Structure of Family Y
 The N-terminal of this group contains the catalytic core of the palm, fingers, and thumb.
 They also have a C-terminal which has a conserved tertiary structure of a four-stranded beta-sheet
supported on one side by two-alpha helices, which are also known as little finger domain. They play a
role in DNA binding and it is essential for complete polymerase activity.
 this family lacks flexibility, unlike the other families.
TYPES OF DNA POLYMERASE
 the types of DNA polymerase are also divided depending on the organism that posses them

eukaryotic and prokaryotic DNA polymerases

Eukaryotic DNA polymerase types

Polymerase γ
 Polymerase γ is a Type A polymerase, whose main function is to replicate and repair
mitochondrial DNA.
 It also functions by proofreading 3′ to 5′ exonuclease activity.
Polymerase α, Polymerase δ, and Polymerase ε
These are the type B Polymerase enzymes and they are the main polymerases applied in
DNA replication.
Pol α works by binding to the primase enzyme, forming a complex, where they both play a
role in initiating replication.
Pol δ starts the synthesis of the lagging strand from Pol α
Pol ε is believed to synthesize the leading strand during replication.
Eukaryotic DNA polymerase types
Studies indicate that Pol δ replicates both the lagging and leading strand.

Pol δ and ε also have a 3′ to 5′ exonuclease activity.

Polymerase β, Polymerase μ, and Polymerase λ

These are type 3 or Family X of polymerase enzymes.

Pol β has a short-patch base excision repair mechanism where it repairs alkylated or oxidized bases.

Pol λ and Pol μ are important for rejoining DNA double-strand breaks due to hydrogen peroxide and
ionizing radiation, respectively.
Eukaryotic DNA polymerase types
Polymerases η, Polymerase ι, and Polymerase κ
They are type 4 or family Y polymerases majorly used in repairing of DNA by a mechanism
known as translesion synthesis.
They are prone to errors during DNA synthesis.
Pol η functions by accurately ensuring the translesion synthesis of DNA damages that is caused
by ultraviolet radiation.
Pol κ is still understudies but one of its known functions is to extend or insert specific bases at
certain DNA lesions.
Translesion synthesis polymerases are activated by stalled replicative DNA polymerase.
Eukaryotic DNA polymerase types
Terminal deoxynucleotidyl transferase (TdT)
TdT functions by catalyzing the polymerization of deoxynucleoside triphosphate to the 3′-
hydroxyl group of a preformed polynucleotide chain.
TdT is a non-template directed DNA polymerase.
It was first detected in the thymus gland.
Prokaryotic DNA polymerase types

DNA Polymerase I
This is a type A or Family A polymerase enzyme that was initially isolated from E. coli and most
abundantly found in E. coli.
Its main function is excision repair of DNA strands from the 3′-5′ direction to the 5′-3 direction,
as an exonuclease.
helps with the maturation of Okazaki fragments
Its role during replication is the addition of nucleotide at the RNA primer and it moves along
the 5′-3′ direction.
The binding site for DNA polymerase I is known as octylglucoside.
 Prokaryotic DNA polymerase types

DNA Polymerase II
It belongs to Type B or Family B of the polymerases.
major function is the 3′ – 5′ exonuclease activity
The DNA polymerase II is found in the replication fork, to help in directing the activities of other polymerases.
DNA Polymerase III
This is the primary enzyme that is used in DNA replication, belonging to the Family C or Type C.
It is responsible for the synthesis of new strands by adding nucleotides to the 3′- OH group of the primer.
It has a 3′-5′ exonuclease activity hence it can also proofread the errors that may arise during DNA strand
replication.
Prokaryotic DNA polymerase types

DNA Polymerase IV
It belongs to the Family Y and it is involved in the non-targeted mutagenesis
stalling activity of the replication fork.
it creates a checkpoint, stops replication, and gives time to properly repair
lesions in the new DNA strand.
 involved in the repair mechanism of translesion synthesis.
It does not have nuclease activity, therefore it is prone to errors in DNA
replication.
Prokaryotic DNA polymerase types

DNA Polymerase V
o Family Y, with high regulatory activity.
oIt is produced only when DNA is damaged and it requires translesion synthesis.
oIt also lacks exonuclease functions and hence it can not proofread the synthesis of DNA replicas
making it less efficient.
Taq DNA polymerase
oTaq polymerase is a thermostable type of DNA polymerase 1 that was initially isolated from a
thermophilic eubacterial known as Thermus aquaticus.
oused in Polymerase Chain Reaction to amplify short strands of DNA.
Mechanism of DNA polymerase

oThe reaction is phosphoryl group transfer.

oThe 3’-OH group of the growing strand acts as a nucleophile and attacks the incoming
deoxyribonucleoside triphosphate at the 𝜶-phosphorus,
oleading to phosphodiester bond formation.
oInorganic phosphate is released in the reaction.
oAll the DNA polymerases require two Mg ions at the active site. 
o DNA polymerase can only add nucleotides at the 3′ end of the growing strand,
o replication always occurs in the 5’→3’ direction.
ocannot initiate the formation of new DNA.
oThey need a template strand, which guides the polymerisation reaction. 
NICK TRANSLATION
1. The nick translation reaction is used to introduce radioactive 
nucleotide phosphates into unlabeled DNA for the purpose of
making a probe.
2. The reaction depends on the ability of the enzyme DNA 
polymerase I to initiate DNA synthesis at free 3´ OH groups, which
are exposed as nicks in the unlabeled DNA. 
3. Nick translation was first developed for labeling 
hybridization probes with radioisotopes
PLASMIDS
•Plasmids are extrachromosomal DNA molecules.
•They are small, circular and have the ability to replicate autonomously.
•Some of the eukaryotes like yeast and plants also contain plasmids.
•Many antibiotic-resistant genes in bacteria are present in plasmids.
•The size of plasmid varies from a few base pairs to thousands of bp.
•Plasmids also get transferred from one bacterial cell to another by the process of conjugation
•Plasmids carrying a specific gene are introduced into bacterial cells, which multiply rapidly 
•Plasmids are used to prepare recombinant DNA with the desired gene to transfer genes from
one organism to another.
PLASMIDS
•Joshua Lederberg coined the term plasmid.
•Plasmids have their own origin of replication (ORI) and they replicate along with the cell so that each
daughter cell possesses a copy of the plasmid also
•Apart from the origin of replication, often it contains genes for antibiotic resistance, for the
production of toxins and other useful genes, that may be required for the survival of cells
Plasmid Vector
•Plasmids and bacteriophages are frequently used as cloning vectors in DNA recombinant technology.
•For genetic engineering purposes, plasmids are artificially prepared in the lab
•The lab-grown plasmids, which are used as a vector contain an origin of replication, cloning site and
selection marker
PLASMIDS
•Origin of Replication (ORI) DNA sequence where initiation of replication starts
•Selectable Marker For selecting bacteria containing desired plasmid, e.g. antibiotic resistance
genes and other specific genes
•Multiple Cloning Sites (MCS) Recognition sites to insert foreign DNA fragment by using
restriction enzymes, a few or single recognition site is preferred to avoid getting several
fragments
•Promoter Region Promotes transcription of the target gene to get the desired protein
•Primer Binding site The sequence of DNA used as a start point for PCR amplification and
sequence verification
 DNA is cut at the specific points by using restriction enzymes (molecular scissors), which make
sticky ends of the DNA
Herbert Boyer and Stanley Norman Cohen together discovered recombinant DNA technology by
recombining DNA segments as desired and inserting them into the bacteria cell to get the desired
protein
The desired genes are then inserted by using DNA Ligase
The recombinant DNA molecule is then introduced to the host bacteria cell by the process
of transformation
The recombinant plasmid then multiplies using host DNA polymerase
The first plasmid used as a cloning vector was pSC101 of Salmonella typhimurium. They showed
that a gene from a frog can be expressed in the bacterial cell
E.coli. plasmid is frequently used as a cloning vector
pBR322 Plasmid

Created in 1977 in the laboratory of Herbert Boyer

The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez."
The main characteristics of pBR322 are:
Restriction sites: BamH I, Hind III, Sal I, Pvu I, Pvu II, Pst I, EcoR I, Cla I
Selectable marker: antibiotic resistance genes for ampicillin (ampR) and tetracycline (tetR)
ORI: the origin of replication
ROP: It codes for proteins, which are involved in the process of replication of plasmid
pBR322 Plasmid
Ti Plasmid

The Tumour inducing or Ti plasmid is present in the bacterium Agrobacterium tumifaciens.

It is widely used now as a cloning vector to deliver desirable genes to the host plant to
get transgenic plants.
Size of the plasmid is ~ 250kbp
There are different kinds of Ti plasmids based on the different genes they possess, which code
for different opines, e.g. leucinopine, nopaline, octopine, etc.
It is a pathogenic species to many dicotyledonous plants. It causes crown gall disease in plants.
It contains one or more T-DNA region
Ti Plasmid

Agrobacterium tumifaciens has the ability to transform the normal cells into tumour cells by
inserting a DNA piece known as T DNA and it starts producing chemicals, that are required by
the bacterium
After inserting the desired gene into Ti plasmid, it loses its pathogenic ability but is still able to
insert the desired gene into the plant cell
It contains vir or virulence genes, which transfer T-DNA region to plant cells and gets integrated
into the plant genome
Ti plasmid can be modified as per the requirement to insert the desired genes
Agrobacterium tumifaciens is called “nature’s genetic engineer”
Ti Plasmid